This invention relates to assays for determining the character of compositions containing heterologous proteins or polypeptides made by recombinant cells. More particularly, this invention is concerned with aiding in predicting the likelihood that a recombinant product will be immunogenic in animal recipients.
It now is known that certain recombinantly-prepared proteins, e.g. human growth hormone (hereinafter hGH) or alpha interferon (.alpha.-IFN) are capable of raising or inducing antibodies in recipients that cross-react with the same protein from nonrecombinant sources. For example, see P. Trown et al., "The Lancet" Jan. 15, 1983: 81-84. It has been speculated that such immunogenicity is the result of aberrant folding of the recombinant protein, protein modifications or denaturation during purification, or the presence of different or extra amino acid residues in the sequence of the recombinant protein. While the immunogenicity of recombinant proteins remains unassociated with adverse clinical consequences in recipient patients, it would be desirable to be able to assay candidate therapeutic compositions for potential immunogenicity in animals. At the present time the only technique available for assessing immunogenicity in humans is to administer the candidate compositions to primates and assay the test animal sera over a period of months to determine whether or not the composition is immunogenic. Not only is this quite expensive, as primate colonies in the United States are limited and in high demand for scientific studies, but considerable time is needed to detect an immunogenic response. Further, so few animals typically are available that it is difficult to assemble a sufficient amount of data.
One of the purposes of immunogenicity studies is to evaluate alternative purification or preparation methodologies for therapeutic proteins. For example, it might be desired to synthesize a recombinant protein as an amino acid sequence variant in which an additional N-terminal methionyl residue is present. Such variants generally result from direct recombinant expression of a protein not having a host-recognized signal sequence. The progress of research will be greatly retarded if clinical trials are needed to evaluate such variants to determine if they are immunogenic. Therefore a significant need exists in the art for an inexpensive and rapid assay that will provide at least some indication as to the likely immunogenicity in humans or other animals of a candidate recombinant product.
On the other hand, it may be desirable to prepare a protein in recombinant culture that is capable of raising antibodies which will cross-react with the protein as it is found in nature. Such recombinant proteins typically are fusions of bacterial proteins with mammalian proteins against which it is desired to stimulate an immune response. The antibodies so produced are useful, for example, in diagnostic assays. The search for highly immunogenic proteins is essentially random. A method is needed for identifying classes of such proteins without the need for in vivo immunization studies.